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Author ManuscriptAuthor Manuscript Author Biochem Manuscript Author Soc Trans. Author Manuscript Author manuscript; available in PMC 2018 February 28. Published in final edited form as: Biochem Soc Trans. 2016 December 15; 44(6): 1659–1666. doi:10.1042/BST20160221.

Motility and adhesion through type IV pili in Gram-positive

Kurt H. Piepenbrink1 and Eric J. Sundberg1,2,3 1Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A 2Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A 3Department of and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A

Abstract Type IV pili are hair-like bacterial surface appendages that play a role in diverse processes such as cellular adhesion, colonization, twitching , formation, and . These extracellular fibers are composed exclusively or primarily of many copies of one or more proteins, tightly packed in a helix so that the highly hydrophobic amino-terminus of the pilin is buried in the core. Type IV pili have been characterized extensively in Gram-negative bacteria, and recent advances in high-throughput genomic sequencing have revealed that they are also widespread in Gram-positive bacteria. Here, we review the current state of knowledge of type IV pilus systems in Gram-positive bacterial species and discuss them in the broader context of eubacterial type IV pili.

Introduction Pili or fimbriae are hair-like appendages present on the surface of bacterial cells. They are universally oligomeric fibers composed of protein subunits called . They can be assembled noncovalently through β-strand insertion (chaperone–usher pili or the recently characterized Bacteroidia FimA family) or through subunit interactions (curli or type IV pili) or covalently through linkage ( wall-linked pili) [1–4]. In type IV pili, the pilin subunits are arranged in a repeating helical pattern. Each pilin consists of an N-terminal helical domain composed primarily of hydrophobic amino acids (bearing a strong resemblance to a transmembrane helix) and a soluble ‘headgroup’ domain. In the assembled pilus, the subunits are arranged such that the hydrophobic N-termini are in the interior of the fiber with the soluble headgroups forming the exterior [5,6]. The noncovalent association of pilin N-termini provides the energy for pilus assembly and the soluble portions are monomeric [5].

Correspondence: Eric J. Sundberg ([email protected]). Competing Interests The Authors declare that there are no competing interests associated with the manuscript. Piepenbrink and Sundberg Page 2

Type IV pili serve a variety of functions including motility along solid surfaces [7,8], Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author adhesion to eukaryotic host cells [9,10], microcolony/biofilm formation [11–13], and horizontal gene transfer [14]. All of these functions are dependent on one or more of three basic activities: (i) extension, lengthening the pilus through polymerization; (ii) adhesion, the ability of one or more pilus subunits to bind to target surfaces or biomolecules; and (iii) retraction, shortening the pilus through depolymerization. For example, , across smooth, dry solid surfaces, involves all three — pili extend, bind weakly to the surface, and as the pilus is retracted, the bacterium is pulled toward the point of attachment.

Type IV pili in Gram-negative bacteria have been divided historically into two classes, types IVa and IVb. This classification is based on evolutionary relationships inferred from the length of an N-terminal signal peptide (the prepilin leader sequence), which is shorter for type IVa pilins, and the identity of the first residue of the mature protein, phenylalanine for type IVa with another hydrophobic residue for type IVb. Type IVa pilins are typically smaller than their type IVb counterparts, forming thinner pilus fibers, and there is less variation in genetic organization among the type IVa pilus systems than their type IVb counterparts [4]. Functionally, type IVa pili are frequently implicated in eukaryotic [9,10] and horizontal gene transfer [15] and less frequently in biofilm formation [11,12], whereas type IVb pili promote bacterial self-association (i.e. microcolony formation or auto-aggregation) [16,17].

Despite the evolutionary gulf between Gram-positive and Gram-negative bacteria, many of the components in the type IV pilus system appear to be conserved; the questions before us in this review are (i) what can we learn from the differences between type IV pili in Gram- positive and Gram-negative bacteria? and (ii) how are diverse functions accomplished through the flexible molecular architecture of type IV pili?

Type IV pilus biogenesis While type IV pili are composed almost entirely of repeating units of a single protein, called either simply the pilin or the major pilin, other proteins, commonly referred to as minor pilins, can be incorporated into the pilus in smaller numbers. Additionally, there are several intracellular proteins required for pilus assembly, extension, and retraction that are typically the most conserved components of type IV pilus systems.

Pilin proteins The three type IV pilus systems highlighted in the figures of this review were chosen as representatives of different classes of type IV pili; pneumoniae R6 contains a Com operon, a DNA-uptake system found in a wide range of Gram-positive and Gram- negative bacteria [18], Clostridium difficile R20291 produces classical type IV pili similar, in some respects, to those of type IVb pilus systems [19,20], and Ruminococcus albus 8 uses a largely uncharacterized and unusual pilus system to adhere to cellulose [21,22]. Also discussed here are unique type IV pilus systems from related bacterial species Clostridium perfringens and Streptococcus sanguinis.

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Figure 1A shows the putative pilin genes for C. difficile R20291, S. pneumoniae R6, and R. Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author albus 8. Each of these genes was identified based on three criteria: (i) the presence of a signal peptide, (ii) a recognition site for a prepilin peptidase, GFxxxE (see below), and (iii) a transmembrane-like α-helix in the predicted protein product. The known major pilin genes (pilA1 for C. difficile and R. albus, comGC for S. pneumoniae; colored orange) are the closest putative pilin genes to their common promoters, consistent with their higher expression levels. Both C. difficile and C. perfringens contain multiple clusters of type IV pilin genes as well as multiple copies of genes encoding pilus biogenesis proteins PilB, PilC, and PilM. The presence of multiple pilB genes in particular implies that Clostridia produce more than one type IV pilus or homologous system, as is the case for several strains of as well as S. sanguinis [23–25]. For C. difficile, only those genes predicted to be involved in the production of PilA1 pili are included in Figure 1.

The genes colored blue (pilK for C. difficile and comGG for S. pneumoniae) likely encode initiator pilins that form the template upon which polymerization begins. These proteins lack a conserved glutamate residue at position 5 of the mature protein, which is thought to form a salt-bridge with the N-terminus of the previously incorporated pilin (and hence is not required for the first subunit) [26], and are much larger than their cognate major pilin proteins. Proteins with these two features can be found in most type IV pilus systems and appear analogous to GspK of the E. coli type II secretion system [27]. EGC03637.1 from R. albus 8 and BAB81985.1 from C. perfringens str. 13 are similarly large and may represent equivalent proteins.

The role of the other genes encoding putative pilins, depicted in gray in Figure 1A, remains to be determined, but we expect that they can be conceptually divided into two classes: those which form a complex with the initiator pilin before the major pilin is polymerized and hence are found primarily at the tip of the pilus and those which are incorporated sporadically throughout the pilus in place of one of the major pilin subunits. The latter category, which we refer to as ‘intercalated pilins’ in Figure 1C, is only sparsely characterized at present and presumably provides no benefit in terms of stabilization; one possible function then is to provide additional adhesive activities to pili into which they are incorporated.

Prepilin processing Any protein that can be incorporated into a type IV pilus is first incorporated into the plasma membrane by the hydrophobic α-helix conserved among all pilins. This insertion is dependent on a signal peptide at the N-terminus [28]. However, before extraction from the membrane and insertion into the pilus, the signal peptide must be cleaved by a prepilin peptidase (Figure 1C). Although poorly conserved, prepilin peptidase genes can be identified clearly in the genomes of C. difficile R20291, S. pneumoniae R6, and R. albus 8 as shown in Figure 1B, as well as in C. perfringens str 13 (WP_043013013.1) and S. sanguinis 2908 (CEL91498.1). Notably, all strains of C. difficile contain two genes encoding putative prepilin peptidases, one of which, pilD2, is similar to the pilD gene of C. perfringens with the other, pilD1 (which is listed in Figure 1B), being more similar to the pilD genes of Gram-negative bacteria [23].

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Pilus extension and retraction Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author All type IV pilus and type II secretion systems utilize cytoplasmic hexameric AAA+ family ATPases to extend the pilus or pseudopilus, respectively. These proteins are universally required for pilus biogenesis and easily identifiable by their sequence similarity. They are typically denoted as PilB (type IV pili), GspE (type II secretion), or ComGA (competence pili). WP_011010862.1 of C. perfringens and CEL91506.1 of S. sanguinis are the equivalents to those PilB proteins listed in Figure 1C. Additionally, an integral membrane protein is also required, typically called PilC, GspF, or ComGB (WP_003451114.1 in C. perfringens and CEL91511.1 in S. san-guinis). How the extension ATPase and the integral membrane protein interact to extend the pilus beyond the plasma membrane remains an area of intense investigation.

In addition to the extension ATPases, many but not all type IV pilus systems include additional related hexameric ATPases (commonly denoted PilT and/or PilU) that are not required for pilus biogenesis and extension but rather for retraction [29]. Correspondingly, ΔpilT mutants are typically hyperpilated as the pili extend but do not retract [30]. Putative retraction ATPases can be found in C. perfringens (WP_003451114.1), C. difficile, R. albus, and S. sanguinis (CEL91511.1) but not in S. pneumoniae (Figure 1B). Competence pili [sometimes referred to as type IV filaments rather than type IV pili] in other organisms also lack pilT homologs raising the obvious question of how DNA bound by these appendages is taken up by the cell. However, we note that some bacteria without pilT genes are nevertheless capable of retracting their pili [31,32]. The mechanism for this retraction remains uncharacterized but given the homology between PilB and PilT, it is not unreasonable to propose that an ancestral ATPase was capable of facilitating both extension and retraction, and that some AAA+ ATPases retain the ability to do so.

Known functions of type IV pili The basic architecture of type IV pili is used for a wide variety of functions including secretion [33], DNA uptake [34], surface motility [14], eukaryotic cell adhesion [10], microcolony formation [35], and even electrical conductance [36] and, in , flagellar motility [37]. We expect, therefore, that in Gram-positive bacteria, the functions of type IV pili will be similarly broad. To date, we can divide the known functions into two classes, adhesion and motility.

Adhesion With the possible exception of the electrically conductive nanowires of Geobacteraceae, all type IV pili have some adhesive activity. Each of the bacterial species discussed here utilizes type IV pili for adhesion in a different fashion; for bacterial self-association, adherence to abiotic surfaces or DNA-binding (shown in Figure 2).

The formation of microcolonies through bacterial self-association, as a prerequisite for the formation of biofilm or other bacterial aggregates, is a well-characterized function of type IVb pili in enteropathogenic E. coli, enterotoxigenic E. coli, and [17,32,38]. The type IVa pili of have also been suggested to play a role in

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biofilm formation by promoting the initial attachment of bacteria [39], and the type IVa pili Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author of gonorrhoeae have been shown to form microcolonies on host cell surfaces in a type IV pilus-dependent manner [40,41].

Varga and colleagues demonstrated that type IV pili were required for native levels of biofilm formation by C. perfringens and more recently, the type IV pili of C. difficile have been also been shown to mediate bacterial autoaggregation in static liquid cultures and microcolony formation as part of in vitro biofilm growth [42–45]. The potential ability of C. difficile to form in vivo may explain the high rate of recurrence of C. difficile infections as in vivo biofilms have been shown to form reservoirs of drug-resistant bacteria that can subsequently expand [46]. The mechanism by which an increase in biofilm occurs remains unclear, but two possibilities are shown in Figure 2A: (i) in the cell-attachment model, type IV pili bind to bacterial surface proteins to form a web that prevents cells from moving independently and (ii) in the pilus bundling model, individual type IV pili from neighboring cells interact with each other from bundles that tie their respective cells together. Additionally, the major pilin of the C. perfringens type IV pili, PilA2, has been shown to mediate adhesion to host eukaryotic cells when expressed by N. gonorrhoeae [47], raising the possibility that the same is true for other infectious clostridial species, including C. difficile, C. tetani, and C. botulinum.

Type IV pili mediate adhesion to abiotic surfaces both in a semi-permanent manner [48] and transiently as part of twitching motility (see below). Rakotoarivonina et al. [21] demonstrated that in R. albus, type IV pili directly mediate the attachment to crystalline cellulose (Figure 2D). It remains unclear whether the major pilin subunit (PilA1 or GP25) is the adhesin, but immunogold microscopy demonstrated that cellulose was able to bind along the entire length of the pilus rather than simply at the tip.

Finally, the type IV pili of S. pneumoniae have been shown to directly bind DNA [49] (Figure 2B). Type IV pili are also required for DNA uptake in many Gram-negative bacteria, and in the case of , a minor pilin, ComP, has been shown to bind DNA directly [50,51]. It remains unclear whether the mechanism of DNA uptake is the same for type IVa pili in N. meningitidis and as it is for the Gram-positive competence pili of S. pneumoniae and subtilis. Notably, the retraction ATPase PilT (which has no homolog in S. pneumoniae) is required for DNA uptake by [14,34].

Motility In the two most widely studied Gram-positive type IV pilus systems, those of C. difficile and C. perfringens, mutants lacking type IV pili have been shown to be deficient in some form of surface motility. Purcell et al. [44] demonstrated that type IV pili mediate twitching motility in C. difficile in a manner dependent on the extension ATPase PilB1; particularly on agar firmer than that typically used for twitching motility assays (1.8% vs. 1.0%; Figure 2C). Varga et al. [52] demonstrated that in C. perfringens, gliding motility (which occurs at an agar–air interface rather than an agar/plastic interface) is dependent on type IV pili with ΔpilC and ΔpilT mutants being nonmotile.

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Like S. pneumoniae, S. sanguinis is naturally competent and contains a homologous type IV Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author pilus system (see above). In addition, S. sanguinis has recently been shown to produce a more conventional type IV pilus system, capable of twitching motility [24,53]. In particular, Gurung et al. note that while ΔpilT mutants are nonmotile, the deletion of a gene of unknown function, pilK, results in cells that are pilated and motile but move only perpendicularly to the long axis of the bacterial chains (rather than along it in a ‘train-like’ motion as the wild-type cells do). Unusually, two major pilin proteins are expressed, PilE1 and PilE2; it remains unclear whether these proteins form separate homopolymers or are combined into a single heteropolymer.

Type IV pilin structure There are many high-resolution structures available for the soluble portion of both major and minor type IV pilins from Gram-negative bacteria. Recently, the structures of two pilin proteins from C. difficile have been resolved [19,20], allowing us to compare type IV pilins from a Gram-positive bacterium with their counterparts from Gram-negative species.

With a single exception of the only available structure of an initiator pilin [31], all known high-resolution structures of the soluble portion of type IV pilins from Gram-negative bacteria are homologous. They show a single domain consisting of a β-sheet sandwiched against an α-helix with a loop of varying length (the αβ loop) connecting the two. The type IV pilins from Gram-negative bacteria also universally contain disulfide bonds, commonly, though not exclusively, at the C-terminus [54–56]; the loop bounded by a C-terminal disulfide bond in a type IV pilin is termed the D-loop.

The structure of PilA1, the C. difficile major type IV pilin, shows a similar overall fold in spite of its lack of sequence similarity (the closest known homolog, TcpA from V. cholerae, has a sequence identity of 14%). Notably, PilA1 lacks cysteine residues and, in the place of the C-terminal disulfide bond typically found in type IV pilins, PilA1 from C. difficile R20291 and NAP08 strains form an antiparallel β-sheet, which we hypothesize increases stability in a similar manner (Figure 3A). However, a recently discovered divergent strain, C. difficile CD160, exhibits an alternate conformation for one of the β-strands, preventing it from forming a β-sheet. Instead, a network of water-mediated hydrogen bonds between the two strands takes the place of the direct hydrogen bonds found in the R20291/NAP08 β- sheet (Figure 3B). These novel stabilization strategies suggest that type IV pilins from Gram-positive organisms may show a greater variability in their C-terminal structures than those from Gram-negative bacteria.

In contrast with the conservation between PilA1 and TcpA, the fold of the minor pilin PilJ is markedly distinct, containing two domains. The first domain contains the typical pilin α- helix at the N-terminus, with a very helical αβ-loop followed by a two-stranded β-sheet (pilins and pseudopilins typically have at least three strands in their central β-sheet). A loop then leads into the second domain that has another helix at a ~70° angle to the N-terminal domain α-helix, followed by a five-stranded β-sheet (Figure 3C). PilJ is the first pilin shown to exhibit this two-domain fold, although there are many similarly sized pilins including CPE2280 from C. perfringens, implying that it may be the first example of a broader class of

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similar minor pilins. This unique combination of two pilin-like folds (Figure 3D) suggests a Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author gene-duplication event, although there is no sequence similarity between the N- and C- terminal domains [19]. Although the function of PilJ remains to be determined, the presence of a second pilin domain that is superfluous for pilus formation strongly implies that it serves an adhesive function, and that this dual-pilin fold is a mechanism by which novel adhesive molecules can be introduced into type IV pili.

Conclusion Despite the ~2 billion years of evolution that separate Gram-negative and Gram-positive bacteria, the structure of the type IV pilus is remarkably well conserved. The strength of the type IV pilus architecture rests on its stability, ease of extension and retraction, and perhaps most importantly, ability to incorporate multiple subunits, which allow a single appendage to perform such distinct functions. However, despite nearly 40 years of study, basic mechanisms of pilus biogenesis and DNA uptake remain mysterious. The study of type IV pili in Gram-positive bacteria provides the opportunity to examine the structure and function of type IV pili in a new context, the potential to shed light on fundamental mechanisms conserved throughout eubacteria, and the possibility of structure-based rational design of novel narrow-spectrum drugs targeting Gram-positive pathogens.

Acknowledgments

Funding

This work was supported by the National Institutes of Health [NIH R01 AI114902] (to E.J.S.). K.H.P. has received direct support from the National Institutes of Health [T32 AI095190], [NIH F32 AI 110045], and [NIH K22 AI123467].

References 1. Busch A, Waksman G. Chaperone-usher pathways: diversity and pilus assembly mechanism. Philos Trans R Soc Lond B Biol Sci. 2012; 367:1112–1122. DOI: 10.1098/rstb.2011.0206 [PubMed: 22411982] 2. Xu Q, Shoji M, Shibata S, Naito M, Sato K, Elsliger MA, et al. A distinct type of pilus from the . Cell. 2016; 165:690–703. DOI: 10.1016/j.cell.2016.03.016 [PubMed: 27062925] 3. Spirig T, Weiner EM, Clubb RT. Sortase enzymes in Gram-positive bacteria. Mol Microbiol. 2011; 82:1044–1059. DOI: 10.1111/j.1365-2958.2011.07887.x [PubMed: 22026821] 4. Giltner CL, Nguyen Y, Burrows LL. Type IV pilin proteins: versatile molecular modules. Microbiol Mol Biol Rev. 2012; 76:740–772. DOI: 10.1128/MMBR.00035-12 [PubMed: 23204365] 5. Craig L, Taylor RK, Pique ME, Adair BD, Arvai AS, Singh M, et al. Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin. Mol Cell. 2003; 11:1139–1150. DOI: 10.1016/S1097-2765(03)00170-9 [PubMed: 12769840] 6. Craig L, Volkmann N, Arvai AS, Pique ME, Yeager M, Egelman EH, et al. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol Cell. 2006; 23:651–662. DOI: 10.1016/j.molcel.2006.07.004 [PubMed: 16949362] 7. Whitchurch CB, Hobbs M, Livingston SP, Krishnapillai V, Mattick JS. Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. Gene. 1991; 101:33–44. DOI: 10.1016/0378-1119(91)90221-V [PubMed: 1676385]

Biochem Soc Trans. Author manuscript; available in PMC 2018 February 28. Piepenbrink and Sundberg Page 8

8. Bradley DE. A function of Pseudomonas aeruginosa PAO polar pili: twitching motility. Can J Author ManuscriptAuthor Manuscript Author Microbiol. Manuscript Author 1980; 26:146–154. Manuscript Author DOI: 10.1139/m80-022 [PubMed: 6105908] 9. Farinha MA, Conway BD, Glasier LM, Ellert NW, Irvin RT, Sherburne R, et al. Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice. Infect Immun. 1994; 62:4118–4123. [PubMed: 7927665] 10. Parker D, Kennan RM, Myers GS, Paulsen IT, Songer JG, Rood JI. Regulation of type IV fimbrial biogenesis in Dichelobacter nodosus. J Bacteriol. 2006; 188:4801–4811. DOI: 10.1128/JB. 00255-06 [PubMed: 16788189] 11. Jurcisek JA, Bakaletz LO. Biofilms formed by nontypeable Haemophilus influenzae in vivo contain both double-stranded DNA and type IV pilin protein. J Bacteriol. 2007; 189:3868–3875. DOI: 10.1128/JB.01935-06 [PubMed: 17322318] 12. Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jørgensen A, Molin S, et al. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol. 2003; 48:1511–1524. DOI: 10.1046/j.1365-2958.2003.03525.x [PubMed: 12791135] 13. Shime-Hattori A, Iida T, Arita M, Park KS, Kodama T, Honda T. Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation. FEMS Microbiol Lett. 2006; 264:89– 97. DOI: 10.1111/j.1574-6968.2006.00438.x [PubMed: 17020553] 14. Harding CM, Tracy EN, Carruthers MD, Rather PN, Actis LA, Munson RS Jr. Acinetobacter baumannii strain M2 produces type IV pili which play a role in natural transformation and twitching motility but not surface-associated motility. mBio. 2013; 4:e00360–13. DOI: 10.1128/ mBio.00360-13 [PubMed: 23919995] 15. Aas FE, Winther-Larsen HC, Wolfgang M, Frye S, Løvold C, Roos N, et al. Substitutions in the N- terminal alpha helical spine of pilin affect type IV pilus assembly, dynamics and associated functions. Mol Microbiol. 2007; 63:69–85. DOI: 10.1111/j. 1365-2958.2006.05482.x [PubMed: 17140412] 16. Anantha RP, Stone KD, Donnenberg MS. Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili. J Bacteriol. 2000; 182:2498–2506. DOI: 10.1128/JB.182.9.2498-2506.2000 [PubMed: 10762251] 17. Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Taylor RK, Levine MM. Toxin, toxin- coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med. 1988; 168:1487–1492. DOI: 10.1084/jem.168.4.1487 [PubMed: 2902187] 18. Imam S, Chen Z, Roos DS, Pohlschröder M. Identification of surprisingly diverse type IV pili, across a broad range of gram-positive bacteria. PLoS ONE. 2011; 6:e28919.doi: 10.1371/ journal.pone.0028919 [PubMed: 22216142] 19. Piepenbrink KH, Maldarelli GA, de la Peña CF, Mulvey GL, Snyder GA, De Masi L, et al. Structure of Clostridium difficile PilJ exhibits unprecedented divergence from known type IV pilins. J Biol Chem. 2014; 289:4334–4345. DOI: 10.1074/jbc.M113.534404 [PubMed: 24362261] 20. Piepenbrink KH, Maldarelli GA, Martinez de la Peña CF, Dingle TC, Mulvey GL, Lee A, et al. Structural and evolutionary analyses show unique stabilization strategies in the type IV pili of Clostridium difficile. Structure. 2015; 23:385–396. DOI: 10.1016/j.str.2014.11.018 [PubMed: 25599642] 21. Rakotoarivonina H, Jubelin G, Hebraud M, Gaillard-Martinie B, Forano E, Mosoni P. Adhesion to cellulose of the Gram-positive bacterium Ruminococcus albus involves type IV pili. Microbiology. 2002; 148(Pt 6):1871–1880. DOI: 10.1099/00221287-148-6-1871 [PubMed: 12055307] 22. Rakotoarivonina H, Larson MA, Morrison M, Girardeau JP, Gaillard-Martinie B, Forano E, et al. The Ruminococcus albus pilA1-pilA2 locus: expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose. Microbiology. 2005; 151(Pt 4):1291–1299. DOI: 10.1099/mic.0.27735-0 [PubMed: 15817796] 23. Melville S, Craig L. Type IV pili in Gram-positive bacteria. Microbiol Mol Biol Rev. 2013; 77:323–341. DOI: 10.1128/MMBR.00063-12 [PubMed: 24006467] 24. Gurung I, Spielman I, Davies MR, Lala R, Gaustad P, Biais N, et al. Functional analysis of an unusual type IV pilus in the Gram-positive Streptococcus sanguinis. Mol Microbiol. 2016; 99:380–392. DOI: 10.1111/mmi.13237 [PubMed: 26435398]

Biochem Soc Trans. Author manuscript; available in PMC 2018 February 28. Piepenbrink and Sundberg Page 9

25. Kulkarni R, Dhakal BK, Slechta ES, Kurtz Z, Mulvey MA, Thanassi DG. Roles of putative type II Author ManuscriptAuthor Manuscript Author Manuscriptsecretion Author and type IV Manuscript Author pilus systems in the virulence of uropathogenic Escherichia coli. PLoS ONE. 2009; 4:e4752.doi: 10.1371/journal.pone.0004752 [PubMed: 19270734] 26. Pasloske BL, Scraba DG, Paranchych W. Assembly of mutant pilins in Pseudomonas aeruginosa: formation of pili composed of heterologous subunits. J Bacteriol. 1989; 171:2142–2147. doi:PMID:2564847. [PubMed: 2564847] 27. Korotkov KV, Hol WGJ. Structure of the GspK–GspI–GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. Nat Struct Mol Biol. 2008; 15:462–468. DOI: 10.1038/ nsmb.1426 [PubMed: 18438417] 28. LaPointe CF, Taylor RK. The type 4 prepilin peptidases comprise a novel family of aspartic acid proteases. J Biol Chem. 2000; 275:1502–1510. DOI: 10.1074/jbc.275.2.1502 [PubMed: 10625704] 29. Merz AJ, So M, Sheetz MP. Pilus retraction powers bacterial twitching motility. Nature. 2000; 407:98–102. DOI: 10.1038/35024105 [PubMed: 10993081] 30. Clemmer KM, Bonomo RA, Rather PN. Genetic analysis of surface motility in Acinetobacter baumannii. Microbiology. 2011; 157(Pt 9):2534–2544. DOI: 10.1099/mic.0.049791-0 [PubMed: 21700662] 31. Kolappan S, Ng D, Yang G, Harn T, Craig L. Crystal structure of the minor pilin CofB, the initiator of CFA/III pilus assembly in enterotoxigenic Escherichia coli. J Biol Chem. 2015; 290:25805– 25818. DOI: 10.1074/jbc.M115.676106 [PubMed: 26324721] 32. Kolappan S, Roos J, Yuen ASW, Pierce OM, Craig L. Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli. J Bacteriol. 2012; 194:2725–2735. DOI: 10.1128/JB.00282-12 [PubMed: 22447901] 33. Peabody CR, Chung YJ, Yen MR, Vidal-Ingigliardi D, Pugsley AP, Saier MH Jr. Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology. 2003; 149(Pt 11):3051–3072. DOI: 10.1099/mic.0.26364-0 [PubMed: 14600218] 34. Wilharm G, Piesker J, Laue M, Skiebe E. DNA uptake by the nosocomial pathogen Acinetobacter baumannii occurs during movement along wet surfaces. J Bacteriol. 2013; 195:4146–4153. DOI: 10.1128/JB.00754-13 [PubMed: 23852865] 35. Tacket CO, Taylor RK, Losonsky G, Lim Y, Nataro JP, Kaper JB, et al. Investigation of the roles of toxin-coregulated pili and mannose-sensitive hemagglutinin pili in the pathogenesis of Vibrio cholerae O139 infection. Infect Immun. 1998; 66:692–695. [PubMed: 9453628] 36. Gorgel M, Ulstrup JJ, Bøggild A, Jones NC, Hoffmann SV, Nissen P, et al. High-resolution structure of a type IV pilin from the metal-reducing bacterium Shewanella oneidensis. BMC Struct Biol. 2015; 15:4.doi: 10.1186/s12900-015-0031-7 [PubMed: 25886849] 37. Jarrell KF, Albers SV. The : an old motility structure with a new name. Trends Microbiol. 2012; 20:307–312. DOI: 10.1016/j.tim.2012.04.007 [PubMed: 22613456] 38. Donnenberg MS, Girón JA, Nataro JP, Kaper JB. A -encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence. Mol Microbiol. 1992; 6:3427–3437. DOI: 10.1111/j.1365-2958.1992.tb02210.x [PubMed: 1362446] 39. O’Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 1998; 30:295–304. DOI: 10.1046/j.1365-2958.1998.01062.x [PubMed: 9791175] 40. Higashi DL, Lee SW, Snyder A, Weyand NJ, Bakke A, So M. Dynamics of Neisseria gonorrhoeae attachment: microcolony development, cortical plaque formation, and cytoprotection. Infect Immun. 2007; 75:4743–4753. DOI: 10.1128/IAI.00687-07 [PubMed: 17682045] 41. Griffiss JM, Lammel CJ, Wang J, Dekker NP, Brooks GF. Neisseria gonorrhoeae coordinately uses Pili and Opa to activate HEC-1-B cell microvilli, which causes engulfment of the gonococci. Infect Immun. 1999; 67:3469–3480. doi:PMID:10377128. [PubMed: 10377128] 42. Maldarelli GA, Piepenbrink KH, Scott AJ, Freiberg JA, Song Y, Achermann Y, et al. Type IV pili promote early biofilm formation by Clostridium difficile. Pathog Dis. 2016; 74doi: 10.1093/ femspd/ftw061 43. Varga JJ, Therit B, Melville SB. Type IV pili and the CcpA protein are needed for maximal biofilm formation by the Gram-positive anaerobic pathogen Clostridium perfringens. Infect Immun. 2008; 76:4944–4951. DOI: 10.1128/IAI.00692-08 [PubMed: 18765726]

Biochem Soc Trans. Author manuscript; available in PMC 2018 February 28. Piepenbrink and Sundberg Page 10

44. Purcell EB, McKee RW, Bordeleau E, Burrus V, Tamayo R. Regulation of type IV pili contributes Author ManuscriptAuthor Manuscript Author Manuscriptto Author surface behaviors Manuscript Author of historical and epidemic strains of Clostridium difficile. J Bacteriol. 2016; 198:565–577. DOI: 10.1128/JB.00816-15 45. Bordeleau E, Purcell EB, Lafontaine DA, Fortier LC, Tamayo R, Burrus V. Cyclic di-GMP riboswitch-regulated type IV pili contribute to aggregation of Clostridium difficile. J Bacteriol. 2015; 197:819–832. DOI: 10.1128/JB.02340-14 [PubMed: 25512308] 46. Kostakioti M, Hadjifrangiskou M, Hultgren SJ. Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harb Perspect Med. 2013; 3:a010306.doi: 10.1101/cshperspect.a010306 [PubMed: 23545571] 47. Rodgers K, Arvidson CG, Melville S. Expression of a Clostridium perfringens type IV pilin by Neisseria gonorrhoeae mediates adherence to muscle cells. Infect Immun. 2011; 79:3096–3105. DOI: 10.1128/IAI.00909-10 [PubMed: 21646450] 48. Giltner CL, van Schaik EJ, Audette GF, Kao D, Hodges RS, Hassett DJ, et al. The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces. Mol Microbiol. 2006; 59:1083–1096. DOI: 10.1111/j.1365-2958.2005.05002.x [PubMed: 16430686] 49. Laurenceau R, Péhau-Arnaudet G, Baconnais S, Gault J, Malosse C, Dujeancourt A, et al. A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae. PLoS Pathog. 2013; 9:e1003473.doi: 10.1371/journal.ppat.1003473 [PubMed: 23825953] 50. Cehovin A, Simpson PJ, McDowell MA, Brown DR, Noschese R, Pallett M, et al. Specific DNA recognition mediated by a type IV pilin. Proc Natl Acad Sci USA. 2013; 110:3065–3070. DOI: 10.1073/pnas.1218832110 [PubMed: 23386723] 51. Berry JL, Xu Y, Ward PN, Lea SM, Matthews SJ, Pelicic V. A comparative structure/function analysis of two type IV pilin DNA receptors defines a novel mode of DNA binding. Structure. 2016; 24:926–934. DOI: 10.1016/j.str.2016.04.001 [PubMed: 27161979] 52. Varga JJ, Nguyen V, O’Brien DK, Rodgers K, Walker RA, Melville SB. Type IV pili-dependent gliding motility in the Gram-positive pathogen Clostridium perfringens and other Clostridia. Mol Microbiol. 2006; 62:680–694. DOI: 10.1111/j.1365-2958.2006.05414.x [PubMed: 16999833] 53. Henriksen SD, Henrichsen J. Twitching motility and possession of polar fimbriae in spreading Streptococcus sanguis isolates from the human throat. Acta Pathol Microbiol Scand B. 1975; 83:133–140. [PubMed: 1171576] 54. Hartung S, Arvai AS, Wood T, Kolappan S, Shin DS, Craig L, et al. Ultrahigh resolution and full- length pilin structures with insights for filament assembly, pathogenic functions, and vaccine potential. J Biol Chem. 2011; 286:44254–44265. DOI: 10.1074/jbc.M111.297242 [PubMed: 22027840] 55. Piepenbrink KH, Lillehoj EP, Harding CM, Labonte JW, Zuo X, Rapp CA, et al. Structural diversity in the type IV pili of multidrug-resistant Acinetobacter. J Biol Chem. 2016 56. Karuppiah V, Collins RF, Thistlethwaite A, Gao Y, Derrick JP. Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis. Proc Natl Acad Sci USA. 2013; 110:E4638–E4647. DOI: 10.1073/pnas.1312313110 [PubMed: 24218553]

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Figure 1. Components of type IV pilus systems (A) Putative pilin genes; major pilins are colored orange, initiator pilins in blue, and all others in gray. Below each gene is the NCBI accession number for the protein it encodes. (B) Type IV pilus biogenesis proteins; accession numbers are listed for PilB, PilT, PilC, and PilD homologs for each species. (C) Type IV pilus biogenesis in Gram-positive bacteria; a schematic showing the proposed organization of type IV pilus proteins in Gram-positive bacteria.

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Figure 2. Functions of type IV pili in Gram-positive bacteria (A) Proposed mechanisms for bacterial self-association through C. difficile type IV pili. (B) DNA-binding by S. pneumoniae type IV pili. (C) Twitching motility in C. difficile. (D) Adhesion to crystalline cellulose by the type IV pili of R. albus.

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Figure 3. Structures of type IV pilins from Gram-positive bacteria (A) Superimposition of C. difficile R20291 PilA1 (blue) with V. cholerae TcpA (gray), panels show the C-terminal disulfide bond of TcpA and β-sheet of PilA1. (B) Comparison of the PilA1R20291/NAP08 β-sheet with the equivalent region of PilA1CD160. (C) Structure of C. difficile PilJ with the N-terminal domain in blue and the C-terminal domain in red and the zinc-binding site. (D) Superimposition of the two PilJ pilin-like domains.

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